Name: studies on the anti-inflammatory effect of xiaoyao pills in the treatment of postmenopausal osteoporosis in mice
Uploaded: 2024-08-23T14:00:00.000+00:00
Duration: 7 min 20 s
Description: Osteoporosis is a common metabolic disease of elderly and postmenopausal women, with no obvious symptoms during its early stages. In the latter stages of ...

百色市科学研究与技术发展计划 （20224128） 为这项工作提供了支持。作者感谢伦敦帝国理工学院的 Dev Sooranna 博士和 YMUN 编辑手稿。YYX 和 ZYW 对这项研究的贡献相同。

小药丸对小鼠绝经后骨质疏松症的治疗作用

作者声明不存在利益冲突。

据统计，骨质疏松症在美国每年导致 150 万例骨折，其中绝大多数发生在绝经后妇女身上 27。随着人口老龄化的增加，预计全球未来大部分髋部骨折将发生在亚洲，到 2050 年，全球髋部骨折总数将达到 820 万例28。预防骨折的成本几乎等于治疗骨折的成本，使用药物不可避免地会产生一些副作用29。因此，迫切需要一种更具成本效益的方法，在?...

世界卫生组织 （WHO） 将骨质疏松症 （OP） 定义为一种以骨量减少和骨组织微结构恶化为特征，导致骨脆性增加，从而增加骨折风险的疾病1。骨质疏松症的临床意义在于它可导致骨折，这与高死亡率、发病率和经济成本有关2。绝经后骨质疏松症 （PMOP） 是由绝经后女性雌激素水平降低引起的，导致破骨细胞活性增加，导致骨质流失和骨微结构破坏。这通常会导致骨质疏松症，对健康产生严重影响3。目前的 PMOP 疗法包括雌激素替代疗法、双膦酸盐和甲状旁腺激素，但它们可能具有不同程度的不良反应、长期依从性不足和高成本4。因此，负担得起的草药对于很大一部分人来说是一种可行的选择。
逍遥丸被列入中国药典5，其中含有八种草药成分，包括柴胡、当归、白芍、白术、茯苓、薄荷草、甘草和新鲜生姜。众所周知，所有这些草药都能有效解肝健脾、养血、调节月经周期，该混合物也用于治疗抑郁症6。然而，逍遥丸在骨质疏松症中的作用尚不清楚。
早期研究表明，炎症会导致骨质流失7 ，并且更年期可能会加速与此过程相关的骨密度下降。此外，骨质疏松症的发展与炎症之间存在密切关系。炎症因子白细胞介素-17 （IL-17） 是由 CD4+ T 淋巴细胞的一个亚群 Th17 细胞分泌的促炎因子。这些细胞与多种慢性炎症有关，它们在类风湿性关节炎骨破坏的发展中起着重要作用8。此外，IL-17 刺激核因子-κ B 配体受体激活剂 （RANKL），RANKL 调节破骨细胞生成，导致比骨形成更大的骨吸收9。IL-17 刺激其他破骨细胞因子的表达，如 TNFα、IL-1、IL-6 和 IL-8。它具有与其他炎症因子协同作用的能力，使其成为重要的炎症效应物10。
研究还表明，逍遥丸与炎症之间存在联系。Shi et al.11 和 Fang et al.12 最近证实，小药丸可以分别降低 IL-6 和 TNF-α 的水平。在另一项代谢相关脂肪性肝炎的研究中，据报道，逍遥丸可以上调丙酸的表达，进而抑制 TNF-α 的表达并发挥抗炎作用13。然而，目前尚不清楚逍遥丸是否可以通过 IL-17 介导炎症反应来调节 PMOM 的发展，这是本研究的目的。
本研究通过网络药理学和生物信息学分析预测了逍遥丸靶点与骨质疏松症相关基因的交集，并分析了蛋白质相互作用、GO 和 KEGG 的交叉基因。基于预测结果，可以观察到 IL-17 信号通路中的关键蛋白 Act1 和 IL-6 的表达14,15，以及骨转换标志物碱性磷酸酶 （ALP） 和 I 型胶原 （COL-1），以观察逍遥丸在 PMOM 小鼠模型中的治疗效果。

<table><tbody><tr><td>1ml Sampler</td><td>Guangxi Beilunhe Medical Industrial Group Co.</td><td>JYQ001</td><td>For anesthesia in mice</td></tr><tr><td>4%polyformaldehyde</td><td>Beijing Solarbio Science &amp;amp; Technology Co.,Ltd.</td><td>P1110</td><td>For tissue fixation</td></tr><tr><td>6-0 absorbable suture (angled needle)</td><td>Shanghai Pudong Jinhuan Medical Supplies Co.</td><td>HZX-06</td><td>For postoperative suturing</td></tr><tr><td>Absorbent cotton ball</td><td>Winner</td><td>MIANQIU-500g</td><td>For sterilization and hemostasis</td></tr><tr><td>Adhesive slides</td><td>Jiangsu Shitai Experimental Equipment Co.</td><td>188105</td><td>For tissue sectioning</td></tr><tr><td>Amobarbital</td><td>Sigma Aldrich (Shanghai) Trading Co.</td><td>A-020-1ML</td><td>For anesthesia in mice</td></tr><tr><td>Bluing Solution</td><td>Beijing Solarbio Science &amp;amp; Technology Co.,Ltd.</td><td>G1866</td><td>Blue coloration of the nuclei of cells after the action of hematoxylin differentiation solution</td></tr><tr><td>C57BL/6 mice</td><td>Beijing Vital River Laboratory Animal Technology Co., Ltd.</td><td>(SCXK 2033-0063)</td><td>For use in animal experiments</td></tr><tr><td>Carbon steel surgical blades</td><td>Premier Medical Equipment Co.</td><td>SP239</td><td>For mouse surgery</td></tr><tr><td>CIKS/TRAF3IP2 Rabbit pAb</td><td>BIOSS ANTIBODIES</td><td>bs-6202R</td><td>Binds to Act1 in tissues</td></tr><tr><td>Cole&#039;s Hematoxylin Solution (For Conventional Stain)</td><td>Beijing Solarbio Science &amp;amp; Technology Co.,Ltd.</td><td>G1140</td><td>For staining paraffin sections</td></tr><tr><td>Collagen Type I Polyclonal antibody</td><td>proteintech</td><td>14695-1-AP</td><td>Binds to COL-1 in tissues</td></tr><tr><td>DAB Substrate kit,20x</td><td>Beijing Solarbio Science &amp;amp; Technology Co.,Ltd.</td><td>DA1010</td><td>For tissue color development</td></tr><tr><td>Disposable surgical sheet 50*60CM</td><td>Nanchang Xuhui Medical Equipment Co.</td><td>SP4529777</td><td>For mouse surgery</td></tr><tr><td>EDTA decalcification solution (pH 7.2)</td><td>Beijing Solarbio Science &amp;amp; Technology Co.,Ltd.</td><td>E1171-500ml</td><td>For tissue decalcification</td></tr><tr><td>Enhanced Endogenous Peroxidase Bloching Buffer</td><td>Beyotime Biotechnology</td><td>P0100B</td><td>Sequestration of tissue or cellular endogenous peroxidases</td></tr><tr><td>Environmentally friendly dewaxing clear liquid</td><td>Servicebio</td><td>G1128-1L</td><td>For dewaxing paraffin sections</td></tr><tr><td>Ethyl Alcohol</td><td>CHRON CHEMICALS</td><td>64-17-5 (CAS)</td><td>For dehydration of paraffin sections</td></tr><tr><td>General Purpose Antibody Diluent</td><td>Epizyme Biotech</td><td>PS119L</td><td>For antibody dilution</td></tr><tr><td>Hematoxylin Differentiation Solution</td><td>Servicebio</td><td>G1039-500ML</td><td>For differentiation after hematoxylin staining and removal of excessively bound and non-specifically adsorbed dye from tissues</td></tr><tr><td>Hematoxylin Eosin (HE) Staining Kit</td><td>Beijing Solarbio Science &amp;amp; Technology Co.,Ltd.</td><td>G1120-3*100ml</td><td>For tissue staining</td></tr><tr><td>High quality stainless steel surgical knife handle</td><td>Premier Medical Equipment Co.</td><td>SP0088</td><td>For mouse surgery</td></tr><tr><td>HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H+L)</td><td>proteintech</td><td>SA00001-2</td><td>Binds to primary antibody and amplifies signal</td></tr><tr><td>IL-17A Polyclonal antibody</td><td>proteintech</td><td>13082-1-AP</td><td>Binds to IL-17 in tissues</td></tr><tr><td>IL-6 Polyclonal antibody</td><td>proteintech</td><td>21865-1-AP</td><td>Binds to IL-6 in tissues</td></tr><tr><td>Immunohistochemistry pen</td><td>Beijing Zhongshan Jinqiao Biotechnology Co.</td><td>ZLI-9305 (YA0310)</td><td>For drawing circles in immunohistochemistry</td></tr><tr><td>Medical surgical suture Non-absorbent (ball) 5-0 3.5m</td><td>Yangzhou Yuanlikang Medical Equipment Co.</td><td>FHX-5-2</td><td>For postoperative suturing</td></tr><tr><td>Medical Suture Needles Angle Needles 4*10 3/8</td><td>Chaohu Binxiong Medical Equipment Co.</td><td>FHZ612-4</td><td>For postoperative suturing</td></tr><tr><td>Neutral Balsam</td><td>Beijing Solarbio Science &amp;amp; Technology Co.,Ltd.</td><td>G8590</td><td>As a slice sealer</td></tr><tr><td>PBS(1X)</td><td>Shenzhen Mohong Technology Co.,Ltd</td><td>B0015</td><td>Buffer for slide washing and partial solution dilution</td></tr><tr><td>Protein Free Rapid Blocking Buffer (1X)</td><td>Epizyme Biotech</td><td>PS108P</td><td>Avoiding non-specific binding of proteins</td></tr><tr><td>Rabbit Anti-Bone Alkaline Phosphatase antibody</td><td>BIOSS ANTIBODIES</td><td>bs-6292R</td><td>Binds to alkaline phosphatase in tissues</td></tr><tr><td>Saline</td><td>Affiliated Hospital Youjiang Medical University For Nationalities</td><td>LHN500</td><td>For animals by gavage</td></tr><tr><td>Shaving/Electric clippers</td><td>HANGZHOU HUAYUAN PET PRODUCTS CO., LTD.</td><td>DTJ-002</td><td>For shaving mice</td></tr><tr><td>Stainless Steel Medical Needle Holder 14cm Coarse Needle</td><td>Premier Medical Equipment Co.</td><td>SP784</td><td>For mouse surgery</td></tr><tr><td>Stainless Steel Ophthalmic Forceps 10.5cm Curved (No Hook)</td><td>Zhuoyouyue</td><td>YKNWW-10.5</td><td>For mouse surgery</td></tr><tr><td>Stainless Steel Ophthalmic Scissors/Surgical Scissors 10CM Straight Tip</td><td>Premier Medical Equipment Co.</td><td>ZYJD-10-ZJ</td><td>For mouse surgery</td></tr><tr><td>Stainless Steel Tip Gastric Needle 12 Gauge 55mm Elbow</td><td></td><td>GWJ-12-55W</td><td>For use in mice by gavage</td></tr><tr><td>Tris-EDTA Antigen Repair Fluid (50x)</td><td>proteintech</td><td>PR30002</td><td>For antigen repair of paraffin sections</td></tr><tr><td>Wooden dissecting board 25*16cm</td><td></td><td>JP16*24</td><td>For mouse surgery</td></tr><tr><td>Xiaoyao pills</td><td>Jiuzhitang Co.,Ltd.</td><td>YPG-041</td><td>For animal drug delivery</td></tr><tr><td>&lt;strong&gt;Others&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>R 4.3.1</td><td></td><td></td><td>Data processing</td></tr><tr><td>Cytoscape3.9.1</td><td>National Resource for Network Biology</td><td></td><td>Building a regulatory network for traditional Chinese medicine</td></tr><tr><td>ImageJ 1.54f</td><td>National Institutes of Health</td><td></td><td>Image processing for immunohistochemistry results</td></tr><tr><td>Adobe Photoshop 24.0.0</td><td>Adobe</td><td></td><td>For image combination</td></tr><tr><td>GraphpadPrism 9.5</td><td>GraphPad Software</td><td></td><td>Statistical analysis of data</td></tr><tr><td>cellsens Dimension</td><td>OLYMPUS</td><td></td><td>For slicing and photographing</td></tr><tr><td>OLYMPUS BX53</td><td>OLYMPUS</td><td></td><td>For HE staining and immunohistochemical section photography</td></tr></tbody></table>

studies on the anti-inflammatory effect of xiaoyao pills in the treatment of postmenopausal osteoporosis in mice

骨质疏松症是老年人和绝经后妇女常见的代谢性疾病，早期无明显症状。在这种情况的后期，患者容易发生骨折，这会严重影响他们的健康和生活质量。全球预期寿命的增加使骨质疏松症成为全球关注的问题。逍遥丹之前 用于治疗抑郁症。此外，该药物似乎具有雌激素样活性，这会影响早期成骨细胞特异性标志物 ALP 和骨细胞外基质主要成分 COL-1 的表达。评估小药丸对小鼠绝经后骨质疏松症 （PMOM） 的影响。通过中医系统药理学 （TCMSP） 数据库获取逍遥丸中各草药成分的靶点信息。利用来自 GeneCards 、 OMIM 、 PharmGkb 、 TTD 、 DrugBank 等网站的信息，通过 Cytoscape 和 String 网络构建草药复合物的调控网络，以评估蛋白质相互作用。对小鼠进行卵巢切除，并用高剂量和低剂量的逍遥丸治疗，并将其与对照组进行比较。通过骨组织的免疫细胞化学评估他们的症状。结果表明，逍遥丸具有通过 IL-17 信号通路缓解去卵巢小鼠 PMOM 症状的能力。这种药物有可能成为治疗骨质疏松症的新型治疗剂。

逍遥丸的活性成分和作用靶点 通过检索 TCMSP 数据库并根据口服生物利用度 （OB） ≥ 30% 和类药性质 （DL） ≥ 0.18 的标准进行筛选，发现小药丸中存在 125 种活性成分。其中，成分 6、4、9、13、2、6、83 和 2 分别来自白芍、白术、薄荷、柴胡、当归、茯苓、甘草和生姜。一些活性成分如表 1 所示。此外，从 TCMSP 数据库中共获得 879 个有?...

作者聚焦：将传统中医与现代药理学和基因组学相结合以评估小鼠绝经后骨质疏松症 

绝经后骨质疏松症已成为一个全球性的公共卫生问题。本研究的目的是探讨中药小药丸对这种情况的治疗效果和相关机制。

逍遥丸治疗小鼠绝经后骨质疏松症的抗炎作用研究

Osteoporosis is a common metabolic disease of elderly and postmenopausal women, with no obvious symptoms during its early stages. In the latter stages of ...

studies-on-anti-inflammatory-effect-xiaoyao-pills-treatment

Research

JoVE Journal

Biology

Studies on the Anti-Inflammatory Effect of Xiaoyao Pills in The Treatment of Postmenopausal Osteoporosis in Mice

424 次观看.  Youjiang Medical University for Nationalities. 本研究的范围是中药逍遥丸对绝经后骨质疏松症小鼠的治疗效果。在这项研究中，我们想确认逍遥丸是否可以发挥治疗效果以及具体机制。在这项研究中，我们将传统中医与现代药理学和基因组学以及关节炎生物信息学相结合，以断言我们所看到的分析将向您展示您的同行。也过程与单一药理学或基因组学相比。分析我们的方案更全面，可能为...

视频: 作者聚焦：将传统中医与现代药理学和基因组学相结合以评估小鼠绝经后骨质疏松症 

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

纯化 M。 magneticum应变AMB - 1磁小体相关蛋白MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

科研

生物学

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

功能成像在小鼠体内的棕色脂肪与FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

人鼻粘膜上皮细胞在气液界面培养

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Pulling Membrane Nanotubes from Giant Unilamellar Vesicles

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many different proteins associate with curved membranes because of their ability to sense or induce membrane curvature. Typically, these processes involve a multitude of proteins making them too complex to study quantitatively in the cell. We describe a protocol to reconstitute a curved membrane in vitro, mimicking a curved cellular structure, such as the endocytic neck. A giant unilamellar vesicle (GUV) is used as a model of a cell membrane, whose internal pressure and surface tension are controlled with micropipette aspiration. Applying a point pulling force on the GUV using optical tweezers creates a nanotube of high curvature connected to a flat membrane. This method has traditionally been used to measure the fundamental mechanical properties of lipid membranes, such as bending rigidity. In recent years, it has been expanded to study how proteins interact with membrane curvature and the way they affect the shape and the mechanics of membranes. A system combining micromanipulation, microinjection, optical tweezers, and confocal microscopy allows measurement of membrane curvature, membrane tension, and the surface density of proteins, concurrently. From these measurements, many important mechanical and morphological properties of the protein-membrane system can be inferred. In addition, we lay out a protocol of creating GUVs in the presence of physiological salt concentration, and a method of quantifying the surface density of proteins on the membrane from fluorescence intensities of labeled proteins and lipids.

Many proteins in the cell sense and induce membrane curvature. We describe a method to pull membrane nanotubes from lipid vesicles to study the interaction of proteins or any curvature-active molecule with curved membranes in vitro.

从巨 Unilamellar 泡中提取膜纳米管

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many ...

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

SA-β-加拉基沙酶筛查检测,用于识别治疗药物

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...

Divergence of Root Microbiota in Different Habitats based on Weighted Correlation Networks

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, which can effectively explore the interaction relationship or co-occurrence model of different microbial species in different environments. The purpose of this manuscript is to provide details on how to use the weighted correlation network algorithm to analyze different co-occurrence networks that may occur in microbial communities due to different ecological environments. All analysis of the experiment is performed in the WGCNA package. WGCNA is an R package for weighted correlation network analysis. The experimental data used to demonstrate these methods were microbial community data from the NCBI (National Center for Biotechnology Information) database for three niches of the rice (Oryza sativa) root system. We used the weighted correlation network algorithm to construct co-abundance networks of microbial community in each of the three niches. Then, differential co-abundance networks among endosphere, rhizoplane and rhizosphere soil were identified. In addition, the core genera in network were obtained by the "WGCNA" package, which plays an important regulated role in network functions. These methods enable researchers to analyze the response of microbial network to environmental disturbance and verify different microbial ecological response theories. The results of these methods show that the significant differential microbial networks identified in the endosphere, rhizoplane and rhizosphere soil of rice.

Network analysis was applied to evaluate the association of various ecological microbial communities, such as soil, water and rhizosphere. Presented here is a protocol on how to use the WGCNA algorithm to analyze different co-occurrence networks that may occur in the microbial communities due to different ecological environments.

基于加权相关网络的不同栖息地根微生物群的差异

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, ...

Dissection of the Endolymphatic Sac from Mice

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the human phenotypes. Although important progress has been done in the understanding of the development and function of the neurosensory epithelia of the cochlea and vestibula, limited knowledge is available regarding the development, cellular composition, molecular pathways and functional characteristics of the endolymphatic sac. This is, in large part, due to the difficulty of visualizing and microdissecting this tissue, which is an epithelium comprised of only one cell layer. The study presented here describes an approach to access and microdissect the endolymphatic sac from the wild-type mouse inner ear at different ages. The result of a similar dissection is shown in a pendrin-deficient mouse model of enlargement of the vestibular aqueduct. A transgenic mouse with a fluorescent endolymphatic sac is presented. This reporter mouse can be used to readily visualize the endolymphatic sac with limited dissection and determine its size. It can also be used as an educational tool to teach how to dissect the endolymphatic sac. These dissection procedures should facilitate further characterization of this understudied part of the inner ear.

<meta charset="utf8"></head><body>小鼠内淋巴囊解剖

This study describes how to dissect the endolymphatic sac from the inner ear of the mouse at different ages. The result of a similar dissection is shown in an Slc26a4-null mouse model of enlargement of the endolymphatic sac. A transgenic mouse with a fluorescent reporter expressed in the endolymphatic sac is presented as a model to readily visualize the endolymphatic sac, and educational tool.

小鼠内淋巴囊解剖

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the ...

逍遥丸治疗小鼠绝经后骨质疏松症的抗炎作用研究

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纯化 M。 magneticum应变AMB - 1磁小体相关蛋白MamAΔ41

功能成像在小鼠体内的棕色脂肪与FDG micro-PET/CT

凋亡细胞清除过程中实时成像

人鼻粘膜上皮细胞在气液界面培养

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

从巨 Unilamellar 泡中提取膜纳米管

SA-β-加拉基沙酶筛查检测,用于识别治疗药物

基于加权相关网络的不同栖息地根微生物群的差异

小鼠内淋巴囊解剖